JP2019533658A - Novel HPV16 non-HLA restricted T cell vaccine, composition thereof and method of use - Google Patents

Abstract

Novel human papillomavirus immunogenic compositions and uses thereof are provided. The composition comprises a unique combination of multi-epitope peptide sequences specifically selected and designed to be effectively processed and cross-presented to T cells. The peptides utilized in the composition exhibit a high level of binding with the HLA supertype. The immunogenic composition is widely applicable to most target populations. The composition includes an adjuvant such as a cationic lipid.

Description

  Embodiments of the present disclosure generally relate to novel HPV16 vaccines, particularly non-HLA restricted T cell vaccines, compositions thereof and methods of use.

  Therapeutic vaccination with HPV E6 and E7 protein antigens provides a strong potential to treat HPV-induced cancers such as cervical cancer, anal cancer, vulvar cancer, vaginal cancer and head and neck cancer and precancerous tumors Has been demonstrated. Because the ability to perform antigen presentation to induce an HPV-specific T cell response is limited, the earliest approach to HPV therapeutic vaccination relies on the inclusion and presentation of a short single CD8 + peptide epitope. It was. HPV vaccines based on these peptides with restricted HLA-A2 epitopes showed very limited applicability even within the selected HLA-A2 population evaluated. Recent approaches to overcome this significant drawback of cancer vaccines are two important approaches or platforms: 1. Delivery of HPV full-length protein-encoding DNA into live vectors such as modified viruses and bacteria, 2. Full-length HPV16 The focus is on the use of multiple overlapping long multi-epitope peptides covering E6 and E7 protein sequences. Both approaches are aimed at overcoming the genetic limitations of patients faced by using short single epitope HLA-A2 peptides to address a broad patient population, and both approaches are human Promising prospects in clinical trials.

  In silico peptide binding analysis has been used efficiently to understand the potential binding capacity of immunogenic peptides. However, this technology has not been used for more efficient cancer vaccine design, which has the characteristics of simplicity and still has the potential to meet the needs of a broad patient population with diverse genetic backgrounds.

  T cells mediate a series of immune responses, including those involved in the delivery of intracellular pathogens, virus-infected cells and tumor cells, and those involved in transplant rejection and autoimmunity. The T cell immune system is adapted to recognize foreign cells as well as modified autologous cells and eliminate them from the body. T cell recognition of peptide antigens occurs via the T cell receptor (TCR). The method requires that peptide antigens are presented to the TCR by major histocompatibility complex (MHC) molecules located on the surface of antigen-presenting cells (APC) such as dendritic cells. Human MHC molecules are referred to as human histocompatibility leukocyte antigen (HLA). The peptide antigen is bound to the MHC molecule in a manner that allows the T cell receptor to recognize the unique structure formed by the combination of the MHC molecule and a specific peptide. A limiting aspect of T cell functionality is that polymorphisms in the MHC molecule, as well as a broad spectrum of unique peptides that can associate with MHC, are only recognized by the function of the T cell clone for a given MHC peptide combination To bring about various recognition patterns.

  There are two types of MHC molecules for antigen presentation, class I and class II. MHC class I molecules consist of an α chain with three domains and a transmembrane and cytoplasmic domain. MHC class I molecules are widely distributed and are present on all nucleated cells. MHC class II molecules include alpha and beta chains that self-associate to form heterodimers. Each chain has two extracellular domains, a transmembrane domain and a cellular domain. MHC class II molecules are more restricted in distribution than class I molecules and are present, for example, on antigen presenting cells (APCs).

  Cytotoxic T lymphocytes ("CTLs") that are specifically activated for a particular antigen can kill cells that contain or express the antigen. The CTL TCR recognizes antigen in association with MHC class I molecules. An important role for T helper lymphocytes (“Th cells”) is the optimal induction of CTL responses, which may also play a role in maintaining CTL memory. Th cell TCRs recognize antigens in the context of MHC class II molecules.

  Therapeutic vaccination to prime antigen-recognizing T cells is a viable option for active immunotherapy of cancer aimed at treating both early and late disease by activating the patient's immune system It has been proven that. The various mechanisms activated by therapeutic vaccination specifically attack antigen-expressing cancer cells and ignore normal cells. Accordingly, cancer treatment vaccines can in principle be effective in suppressing tumor growth and treating recurrent tumors that are refractory to conventional therapies such as surgery, radiation therapy and chemotherapy. Cancer treatment vaccines for treating prostate cancer have already been approved by the US Food and Drug Administration. This major breakthrough has paved the way for a new approach for therapeutic vaccination that may provide improved safety and efficacy. Several such approaches are currently being evaluated preclinically and clinically. Unlike prophylactic antibody-derived vaccines, which are generally administered to healthy individuals, cancer treatment vaccines are administered to cancer patients to eradicate cancer cells by enhancing the patient's own immune response, particularly the T cell response. Designed (Lollini PL, Cavallo F, Nanni P, Forni G. Vaccines for tumour prevention. Nature reviews. Cancer. 2006; 6: 204-216).

Tumor-associated antigens as therapeutic targets Recombinant vaccines based on defined tumor-associated antigen (TAA) -derived proteins, or synthetic peptide vaccines derived from TAA, usually administered in combination with adjuvants or immune modulators, are self-derived And shows significant advantages in cost and simplicity over DC vaccines. The availability of patient samples or specimens and the complex procedures for preparing individualized vaccines limit the widespread use of autologous cancer vaccines. MAGE-1 is the first gene reported to encode a human tumor antigen recognized by T cells (van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B , Knuth A, Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science. 1991; 254: 1643-1647.), And has been well studied and used in clinical cancer vaccines. The identification of several TAAs provided the ability to develop and design a variety of targeted therapeutic vaccines to address a wide range of cancers. Such TAAs are divided into several major categories. Cancer-testis antigens such as NY-ESO-1, BAGE, MAGE, and SSX-2 are normally silenced in adult tissues but are encoded by genes that are transcriptionally reactivated in tumor cells (De Smet C, Lurquin C, van der Bruggen P, De Plaen E, Brasseur F, Boon T. Sequence and expression pattern of the human MAGE2 gene.Immunogenetics. 1994; 39: 121-129; Gnjatic S, Ritter E, Buchler MW, Giese NA, Brors B, Frei C, Murray A, Halama N, Zornig I, Chen YT, Andrews C, Ritter G, Old LJ, Odunsi K, Jager D. Seromic profiling of ovarian and pancreatic cancer.Proceedings of the National Academy of Sciences of the United States of America.2010; 107: 5088-5093; Hofmann O, Caballero OL, Stevenson BJ, Chen YT, Cohen T, Chua R, Maher CA, Panji S, Schaefer U, Kruger A, Lehvaslaiho M, Carninci P, Hayashizaki Y, Jongeneel CV, Simpson AJ, Old LJ, Hide W. Genome-wide analysis of cancer / testis gene expression.Proceedings of the National Academy of Sciences of the United S tates of America.2008; 105: 20422-20427; Karbach J, Neumann A, Atmaca A, Wahle C, Brand K, von Boehmer L, Knuth A, Bender A, Ritter G, Old LJ, Jager E. Efficient in vivo priming by vaccination with recombinant NY-ESO-1 protein and CpG in antigen naive prostate cancer patients. Clinical cancer research: an official journal of the American Association for Cancer Research. 2011; 17: 861-870). Tissue differentiation antigens are antigens derived from normal cells and are shared by both normal cells and tumors, but melanoma (gp100, Melan-A / Mart-1 and tyrosinase) (Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, Figdor CG.Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes.J Exp Med. 1994; 179: 1005-1009..Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, Figdor CG. Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med. 1994; 179: 1005-1009), prostate cancer (PSA) , PAP) (Correale P, Walmsley K, Nieroda C, Zaremba S, Zhu M, Schlom J, Tsang KY. In vitro generation of human cytotoxic T lymphocytes specific for peptides derived from prostate-specific antigen. J Natl Cancer Inst. 1997; 89: 293-300; Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, Redfern CH, Ferrari AC, Dreicer R, Sims RB, Xu Y, Froh Lich MW, Schellhammer PF. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. The New England journal of medicine. 2010a; 363: 411-422) and breast cancer (Mammaglobin A) (Jaramillo A, Majumder K, Manna PP, Fleming TP, Doherty G, Dipersio JF, Mohanakumar T. Identification of HLA-A3-restricted CD8 + T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer.International journal of cancer.Journal international du cancer.2002; 102: 499-506) and other tumor cells are elevated. Like these differentiation-related antigens, CEA (Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J. Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine J Natl Cancer Inst. 1995; 87: 982-990), MUC-1 (Finn OJ, Gantt KR, Lepisto AJ, Pejawar-Gaddy S, Xue J, Beatty PL.Importance of MUC1 and spontaneous mouse tumor models for understanding the Immunobiology of human adenocarcinomas.Immunologic research.2011; 50: 261-268), HER2 / Neu (Disis ML, Wallace DR, Gooley TA, Dang Y, Slota M, Lu H, Coveler AL, Childs JS, Higgins DM, Fintak PA , dela Rosa C, Tietje K, Link J, Waisman J, Salazar LG.Concurrent trastuzumab and HER2 / neu-specific vaccination in patients with metastatic breast cancer.Journal of clinical oncology, official journal of the American Society of Clinical Oncology. 2009; 27: 4685-4692), tumor suppressor gene (p53) (Azuma K, Shichijo S, Maeda Y, Nakatsura T, Nonaka Y, F ujii T, Koike K, Itoh K. Mutated p53 gene encodes a nonmutated epitope recognized by HLA-B * 4601-restricted and tumor cell-reactive CTLs at tumor site.Cancer Res. 2003; 63: 854-858), hTERT (Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity. 1999; 10: 673-679) and certain anti-apoptotic proteins (e.g. survivin) (Vonderheide RH, Hahn WC, Schultze JL, Nadler LM.The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity. 1999; 10: 673-679) Is also elevated in tumor tissue compared to normal corresponding tissue. Unique tumor-specific antigens are often referred to as mutant oncogenes (ras, B-raf) (Brichard VG, Lejeune D. Cancer immunotherapy targeting tumour-specific antigens: towards a new therapy for minimal residual disease Expert opinion on biological therapy. 2008; 8: 951-968). Targeting these tumor-specific antigens involved in promoting the tumorigenesis process has the advantage of being resistant to immune selection and may be more effective. A number of such tumor-specific antigens have been identified and utilized, but considering how a wide variety of genetic profiles among human populations are considered, regardless of how such groups are identified, for example There is a need to identify antigens with increased specificity for a particular group, such as ethnicity or geographic location: For various reasons such as: there is an ever increasing demand for the identification of such additional antibodies. There remains a need in the pharmaceutical industry to more accurately and simplify the process of manufacturing vaccines, and therefore it is simple to simply use the antigens, proteins and peptides most accurately associated with eliciting an appropriate immunological response. It is an ongoing goal.

Protein / peptide-based vaccines are clearly cost-effective over self-derived or individualized vaccines. However, the fact that they target only one epitope or a small number of epitopes of TAA may be considered disadvantageous. It is generally considered that the induction of both antigen-specific CTL and antigen-specific CD4 ⁺ helper T cells is necessary for a cancer vaccine to be optimally effective. Some polypeptide vaccines (eg, Stimuvax®) potentially contain both CD4 and CD8 epitopes. Another approach to enhance the immunogenicity of self-antigens is to modify the peptide sequence of TAA to introduce enhancer agonist epitopes, which increase peptide binding to MHC molecules or T cell receptors Resulting in higher levels of T cell response and / or higher T recell binding activity (Dzutsev AH, Belyakov IM, Isakov DV, Margulies DH, Berzofsky JA. Avidity of CD8 T cells sharpens immunodominance. International immunology. 2007; 19: 497-507; Jordan KR, McMahan RH, Kemmler CB, Kappler JW, Slansky JE.Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens.Proceedings of the National Academy of Sciences of the United States of America 2010; 107: 4652-4657; Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, Restifo NP, Dudley ME, Schwarz SL, Spiess PJ, Wunderlich JR, Parkhurst MR, Kawakami Y, Seipp CA , Einhorn JH, White DE. Imm unologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med. 1998; 4: 321-327).

  A common approach to cancer treatment vaccination is vaccination with the correct MHC human leukocyte antigen (HLA) -binding peptide derived from the sequence of TAA. T cells recognize their target antigens as 8-10 amino acid peptides presented by MHC class I molecules on the cell surface. The main drawback of such an approach is the fact that humans are genetically diverse with a wide range of HLA alleles that recognize and bind to different peptide antigens. As a result, cancer peptides based on short peptides have very limited applicability, and recent approaches have in some cases exceeded 10 peptides with the aim of providing a reasonable range of populations The inclusion of multiple peptides was required.

HPV vaccine
HPV E6 and E7 proteins are the most abundant viral transcripts constitutively co-expressed in all HPV-infected precancerous cells and found in biopsies from HPV-related cervical cancer cells (K. Seedorf, T. Oltersdorf, G. Krammer, W. Rowekamp, Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells.EMBO J. 6, 139-144 (1987) ). For interaction with p53 and retinoblastoma protein (D. Pim, A. Storey, M. Thomas, P. Massimi, L. Banks, Mutational analysis of HPV-18 E6 identifies domains required for p53 degradation in vitro, Oncogene 9, 1869-1876 (1994)), E6 and E7 are involved in cell transformation and are required for the maintenance of HPV-related malignancies. abolition of p53 transactivation in vivo and immortalisation of primary BMK cells. (K. Munger, PM Howley, Human papillomavirus immortalization and transformation functions. Virus Res. 89, 213-228 (2002)). In particular, E6- and E7-specific cellular immune responses are associated with antibodies of HPV16-related lesions (S. Peng, C. Trimble, L. Wu, D. Pardoll, R. Roden, CF Hung, TC Wu, HLA- DQB1 * 02-restricted HPV-16 E7 peptide-specific CD4 + T-cell immune responses correlate with regression of HPV-16-associated high-grade squamous intraepithelial lesions. Clin. Cancer Res. 13, 2479-2487 (2007)). Farhat et al. Found that the proportion of positive enzyme-linked immunospot (ELISpot) responses to HPV16 E6 and E7 in women with recently elucidated HPV infection compared to women with persistent cervical HPV16 infection. (S. Farhat, M. Nakagawa, AB Moscicki, Cell-mediated immune responses to HPV-16 E6 and E7 antigens as measured by interferon gamma enzyme-linked immunospot in women with cleared or persistent human papillomavirus infection. Int. J. Gynecol. Cancer 19, 508-512 (2009)). Thus, HPV E6 and E7 antigens are considered promising immunotherapy targets. To date, several HPV therapeutic vaccines, including protein / peptide-based vaccines (L. Muderspach, S. Wilczynski, L. Roman, L. Bade, J. Felix, LA Small, WM Kast, G. Fascio, V. Marty, J. Weber, A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive.Clin. Cancer Res. 6, 3406-3416 (2000); WJ van Driel, ME Ressing, GG Kenter, RMP Brandt, EJT Krul, AB van Rossum, E. Schuuring, R. OVringa, T. Bauknecht, A. Tamm-Hermelink, PA van Dam, GJ Fleuren, WM Kast, CJM Melief and JB Trimbos, Vaccination with HPV16 Peptides of Patients with Advanced Cervical Carcinoma: Clinical Evaluation of a Phase I-II Trial, Eur J Cancer, Vol. 35, No. 6, pp. 946-952, 1999), HPV E6 and It has been developed with an emphasis on stimulating the production and activation of E7-specific T cells. However, HPV vaccines based on these peptides with restricted HLA-A2 epitopes showed very limited applicability even within the evaluated and selected HLA-A2 population. In 2009, an HPV peptide vaccine containing 13 overlapping peptides from the HPV16 E6 protein and 4 overlapping peptides from the HPV16 E7 peptide, when tested in a non-restricted patient population with VIN3, is a potent anti-HPV It was reported to show a response. This study provided the first demonstration of a widely acting HPV peptide vaccine in a non-HLA restricted population (Gemma G. Kenter, Marij JP Welters, A. Rob PM Valentijn, Margriet JG Lowik, Dorien MA Berends-van der Meer, Annelies PG Vloon, Farah Essahsah, Lorraine M. Fathers, Rienk Offringa, Jan Wouter Drijfhout, Amon R. Wafelman, Jaap Oostendorp, Gert Jan Fleuren, Sjoerd H. van der Burg, and Cornelis JM Melief; Vaccination against HPV- 16 Oncoproteins for Vulvar Intraepithelial Neoplasia, N Engl J Med 2009; 361: 1838-47).

  These references by Kenter et al., Particularly recent publications, highlight the main drawbacks associated with current approaches to the development of peptide vaccines that provide a broad HLA coverage. Current approaches focus on the development of long peptides that cover the entire sequence of the antigen protein, as immunogenic sequences that cannot provide a wide range of coverage cannot be determined. As a result, there are four major drawbacks: 1. Vaccines are complex and involve the use of a large number of peptides, for example 13 peptides in Kenter et al .; 2. Vaccines are more expensive than necessary 3. subject the patient to receive unwanted peptides that may not provide any therapeutic or immunogenic benefit; and 4. competitive binding of the active peptide with other peptides (This particular drawback has been reported by Kenter et al., Supra).

  The present invention reports an efficient approach to the development of simple, more cost-effective and highly effective and extensive HLA-coated peptide vaccines.

Immunostimulatory adjuvants for protein / peptide based vaccines
Given that TAA is inherently poorly immunogenic, an immunostimulatory adjuvant may be necessary in some cases for the development of an effective immune response. Aluminum salts (alum) have been used as adjuvants with great success for almost a century and have been effective in promoting protective humoral immunity. However, alum is least effective against diseases where cellular immunity is required for protection. The recognition over the last 20 years that activation of innate immunity is necessary to promote the adaptive immune response has fundamentally changed the theory of how adjuvants promote adaptive immunity. In particular, Charles Janeway's pioneering work demonstrated that adaptive immune responses precede and depend on innate immune receptors caused by microbial components (Janeway CA., Jr. The immune system evolved to discriminate infectious Nonself from noninfectious self. Immunol Today. 1992; 13: 11-16). Recognition of conserved moieties associated with pathogens or pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors, such as toll-like receptors (TLR), is a coordinated natural and adaptive immunity against microbial pathogens or infectious cells. Involved (Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011; 34: 637-650). TLR-mediated activation of antigen presenting cells (eg, DC) is an important step in this process. In fact, many established experimental vaccines incorporate PAMP not only to protect against infections but also as part of therapeutic vaccination against cancer (Wille-Reece U, Flynn BJ , Lore K, Koup RA, Miles AP, Saul A, Kedl RM, Mattapallil JJ, Weiss WR, Roederer M, Seder RA.Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates. J. Exp. Med. 2006; 203: 1249-1258). The use of these molecularly and functionally defined molecules as adjuvants greatly facilitates the rational design of vaccines.

  In support of this view, BCG (Bacillus Calmette-Guerin), which has been used for many years for the treatment of bladder cancer, is relatively effective and can activate TLR2 and TLR4 Heldwein KA, Liang MD, Andresen TK, Thomas KE, Marty AM, Cuesta N, Vogel SN, Fenton MJ. TLR2 and TLR4 serve distinct roles in the host immune response against Mycobacterium bovis BCG. Journal of leukocyte biology. 2003; 74: 277-286). LPS, a natural ligand of TLR4, was reported to have anticancer properties as early as the 1960s (Mizuno D, Yoshioka O, Akamatu M, Kataoka T. Antitumor effect of intracutaneous injection of bacterial lipopolysaccharide. Cancer research. 1968; 28: 1531-1537). Monophosphoryl lipid A (MPL) is a chemically modified derivative of the endotoxin of S. mnnesota that exhibits very low toxicity but retains most of the immunostimulatory properties of LPS (Mata-Haro V, Cekic C, Martin M, Chilton PM, Casella CR, Mitchell TC. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science. 2007; 316: 1628-1632). Many researchers have shown that MPL strongly enhances the patient's immune response to viral and tumor-associated antigens (Schwarz TF. Clinical update of the AS04-adjuvanted human papillomavirus-16 / 18 cervical cancer vaccine, Cervarix. Advances in therapy. 2009; 26: 983-998). FDA has approved Cervarix vaccine formulated with MPL and aluminum salt as a preventive vaccine against human papillomavirus (Schiffman M, Wacholder S. Success of HPV vaccination is now a matter of coverage. The lancet oncology. 2012; 13: 10 -12). Imiquimod (TLR agonist) was approved by the FDA in 2004 for human use against actinic keratosis and superficial basal cell carcinoma (Hoffman ES, Smith RE, Renaud RC., Jr. From the analyst's couch : TLR-targeted therapeutics. Nat Rev Drug Discov. 2005; 4: 879-880). These TLR agonists have strong potential in promoting the immunogenicity of less immunogenic TAAs. Indeed, several peptide / protein based cancer vaccines in combination with TLR agonists are being tested in clinical trials. These include ampligens targeting TLR3 (NCT01355393), histonor targeting TLR3 (NCT00773097, NCT01585350, NCT01437605), MELITAC 12.1 targeting TLR4 (NCT01585350) and reciquimod targeting TLR9 (NCT00960752) . The family of PRRs has grown greatly in recent years, thus investigating the role of innate immune pathways in defining the mechanism of adjuvant action and the role of other PRRs (eg, NLR, RLR) in the adjuvant activity of cancer therapeutic vaccines A great deal of effort is spent on doing this.

  In addition to sensing pathogen-related signals, PRP also recognizes endogenous “alarmins” such as stress / heat shock protein (HSP) and HMGB-1 (Lotze MT, Zeh HJ, Rubartelli A, Sparvero LJ, Amoscato AA, Washburn NR, Devera ME, Liang X, Tor M, Billiar T. The grateful dead: damage-associated molecular pattern molecules and reduction / oxidation regulate immunity. Immunol Rev. 2007; 220: 60-81; Todryk SM, Melcher AA, Dalgleish AG, Vile RG. Heat shock proteins refine the danger theory. Immunology. 2000; 99: 334-337). As an intrinsic and highly conserved protein component of cells, these damage-related molecular patterns (DAMP) also communicate the nature and magnitude of cytotoxicity to the host immune system. HSP is known to act as a molecular chaperone involved in quality control of intracellular proteins (Calderwood SK, Murshid A, Prince T. The shock of aging: molecular chaperones and the heat shock response in longevity and aging --a mini-review. Gerontology. 2009; 55: 550-558; Mayer MP, Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism.Cell Mol Life Sci. 2005; 62: 670-684), over the past 20 years Studies have established the concept that specific HSPs can integrate both natural and adaptive immune responses and can be used as immunostimulators for immunotherapy (Mayer MP, Bukau B. Hsp70 chaperones: cellular functions Cell Mol Life Sci. 2005; 62: 670-684; Wang XY, Facciponte JG, Subjeck JR. Molecular chaperones and cancer immunotherapy. Handb Exp Pharmacol. 2006b; 172: 305-329).

  While the rational design of vaccines has made significant progress, there is a continuing need for the development of vaccines that are both prophylactic and therapeutically optimized. There is a need for the development of vaccines with broad applicability for large patient populations, and for such vaccines to be specific and effective.

  Disclosed herein is a novel composition comprising an HPV peptide sequence, optionally combined with one or more adjuvants, wherein the HPV peptide sequence is HPV E6 peptide and / or HPV16 E7 peptide And the peptide sequence has a binding affinity of 5 HLA supertypes and an IC50 of about 5000 nM, and some of the peptides are multi-epitope. In certain embodiments, the composition comprises an adjuvant consisting of a cationic lipid, and in certain embodiments, the cationic lipid is DDA, R-DOTAP, DOTAP, DOTMA or DOEPC, variations or similar thereof. Consists of the body. The novel compositions disclosed herein are superior to currently available vaccines in that they are effective in more than 80-90% of the general population.

  Disclosed herein is a method for inducing an immune response against an HPV infection in a subject comprising administering to the subject a composition comprising an HPV peptide sequence, optionally in combination with an adjuvant, wherein The HPV peptide sequence corresponds to HPV16 E6 peptide and / or HPV16 E7 peptide, and the peptide sequence has a binding affinity of 5 HLA supertypes and an IC50 of about 5000 nM, and several The peptide is a multi-epitope. The method can be prophylactic or therapeutic.

FIG. 1 provides graphs showing the response of anti-HPV16 E6 and E7 in interferon-γ assay by subjects and visits by 1 mg and 3 mg R-DOTAP cohorts. Abbreviations: Bkg = background (striped bars); PBMC = peripheral blood mononuclear cells; R-DOTAP = 1,2-dioleoyl-3-trimethylammonium-propane chloride R-enantiomer; SFC = SFU = spot formation Unit; Stim = stimulated (transparent bar). Data represent the mean SFU per 4 × 10 ⁵ PBMC in triplicate wells. Striped bars represent background SFU in wells stimulated with medium alone. Clear bars represent SFU in wells stimulated with peptide pool. Visit 2 = Day 1 before vaccination; Visit 3 = Day 15 after 14 days after vaccination 1; Visit 5 = Day 36 after 14 days after vaccination 2; Visit 7 = 57 after 14 days after vaccination 3 Day 9; Visit 9 = Day 133, 90 days after vaccination 3. FIG. 2 provides a graph showing the response of anti-HPV16 E6 and E7 in an interferon-γ assay by a subject and a visit with a 10 mg R-DOTAP cohort. Data represent the mean SFU per 4 × 10 ⁵ PBMC in triplicate wells. Striped bars represent background SFU in wells stimulated with medium alone. Clear bars represent SFU in wells stimulated with peptide pool. Visit 2 = Day 1 before vaccination; Visit 3 = Day 15 after 14 days after vaccination 1; Visit 5 = Day 36 after 14 days after vaccination 2; Visit 7 = 57 after 14 days after vaccination 3 Day 9; Visit 9 = Day 133, 90 days after vaccination 3. FIG. 3 shows a tumor corresponding to peptides corresponding to SEQ ID NO: 1 (white squares), SEQ ID NOs: 7, 8, 31 32 and 31 (black circles), SEQ ID NO: 7 (white circles), and SEQ ID NO: 8 (black triangles). A sample tumor regression plot is provided that shows the results of the effect of the peptide on the criminally measured median tumor size. In FIG. 3, a composition containing only R-DOTAP is represented by a plot line marked with a striped square.

DETAILED DESCRIPTION OF THE INVENTION The following detailed description is exemplary and explanatory and is intended to provide further explanation of the present disclosure as described herein. Other advantages and novel features are expected to be readily apparent to those skilled in the art from the following detailed description of the disclosure. The references mentioned herein, including US Provisional Patent Application No. 62 / 404,458, are incorporated by reference in their entirety.

  What is disclosed herein is screened and demonstrated in HLA-A2 humanized transgenic mice and effectively processed and cross-presented to T cells as confirmed in various human subjects. A method for the design and use of unique peptide sequences, including unique multi-epitope peptides derived from HPV16 E6 and E7. In certain embodiments, the new composition comprising the new peptide sequence consists of 2-8 peptide sequences, 2-6 peptide sequences, or 4 peptide sequences. The peptide can be incorporated into an immunogenic composition such as a vaccine. As demonstrated below, in silico peptide binding analysis against major HLA supertypes confirms that the resulting compositions and vaccines address over 80-90% of the population addressing. Yes. In non-HLA restricted human clinical trials involving the use of the peptide compositions described herein, strong T cell induction was confirmed in all subjects. Utilizing a novel approach for in silico binding analysis combined with testing in humanized transgenic mice, the inventors first developed a simple, effective and widely applicable peptide-based HPV16 cancer therapeutic vaccine. .

  An important consideration in the design of vaccines based on peptides designed to induce the generation and response of CD8 + T cells in humans is the polymorphism of HLA class I molecules in the population. Because different HLA alleles bind to different peptides, it is important that peptide vaccines contain enough different peptides to be immunogenic in a high proportion of the population. The inventors have designed a novel multi-peptide vaccine in this application to cover the immunogenic region of HPV16 E6 and E7 proteins to provide correct processing and presentation of CD8 + T cell epitopes in humans. In one embodiment, the vaccine comprises four HPV related peptides selected based on their binding and immunogenic activities. As detailed in the Examples, we worked on ELISpot and tumor regression studies and in silico analysis to predict potential HLA alleles that could bind to various HPV peptides. Next, the ability of the human immune system to present and recognize peptide epitopes was examined in humanized transgenic mice to confirm presentation, processing and subsequent induction of antigen-specific CD8 + T cell responses. Finally, the ability to induce human T cell responses was studied in non-HLA-restricted human clinical trials and the ability of formulations to induce strong T cell responses in subjects with various HLA subtypes was confirmed.

  In one embodiment, the novel composition described herein comprises an HPV peptide sequence in combination with an adjuvant, wherein the HPV peptide sequence corresponds to an HPV16 E6 peptide and / or HPV16 E7 peptide, and the peptide sequence Have a binding affinity of 5 HLA supertypes and an IC50 of about 5000 nM. In certain embodiments, the peptide is a multi-epitope. Said peptides may comprise their lipidated versions comprising SEQ ID NO: 5, 9, 10 and 11 or comprising SEQ ID NO: 23, 24, 25 and 26. In one embodiment, the peptides may be present in the composition as individual peptides or spacers to form a single long peptide comprising the claimed sequence by methods known to those skilled in the art. Can be conjugated to each other (in any order) with or without a spacer. In certain embodiments, the HLA supertype is HLA-A * 02: 01, HLA-A * 03: 01, HLA-A * 24: 02, HLA-B * 07: 02, and HLA-B * 58. : 01 is included. In some embodiments, the composition comprises an enhancer agonist epitope such as an HBV core helper peptide, and / or a peptide encoded by SEQ ID NO: 14, or those such as SEQ ID NO: 15 and SEQ ID NOs: 27 and 28 It may further comprise a single epitope peptide comprising a peptide encoded by an analogue of The compositions described herein further include modified peptides, peptide analogs, and active fragments thereof. In certain embodiments, the peptide is known to those skilled in the art for oxidation, disulfide bond crosslinking, pegylation, glycosylation, phosphorylation, palmitoylation, methylation, biotinylation, or to improve efficacy and immunogenicity. It can be modified by other methods. In one embodiment, the adjuvant of the composition comprises a cationic lipid, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R- It can be selected from the group consisting of DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, and variations or analogs thereof. In one embodiment, the novel composition of the present disclosure comprises peptides corresponding to SEQ ID NOs: 5, 9, 10, and 11, the adjuvant comprises a cationic lipid, and the cationic lipid comprises R-DOTAP . In one embodiment, the novel composition of the present disclosure comprises a peptide corresponding to SEQ ID NO: 23, 24, 25 or 26, the adjuvant comprises a cationic lipid, and the cationic lipid comprises R-DOTAP. . In one embodiment, the novel composition of the present disclosure comprises a peptide corresponding to SEQ ID NO: 5, 9, 10, 11, 23, 24, 25 or 26, the adjuvant comprises a cationic lipid, and the cationic Lipids include R-DOTAP. The above embodiments can optionally be encapsulated in liposomes. The above embodiments can be applied to pharmaceutically acceptable carriers and excipients, acetates, phosphates and sucrose, various buffers such as isotonic agents such as trehalose, or surfactants such as tweens and others skilled in the art. Can be arbitrarily combined with known ones.

  In one embodiment, the disclosure herein includes one or more of SEQ ID NOs: 5, 9, 10, 11 or one or more of SEQ ID NOs: 23, 24, 25, or 26 operably linked to a promoter. A recombinant vector comprising a nucleic acid molecule encoding a polynucleotide is provided. One or more of SEQ ID NOs: 5, 9, 10, 11 or one of 23, 24, 25 or 26 operably linked to a promoter further comprising a nucleic acid molecule encoding an enhancer for the epitope and / or single epitope peptide Also provided is a recombinant vector comprising a nucleic acid molecule encoding a peptide comprising the above. In one embodiment, the vector can include a recombinant adenovirus. As is known to those skilled in the art, nucleic acid sequences can be cloned using conventional molecular biology techniques, or synthesized de novo by DNA synthesis, which includes DNA synthesis and / or molecular cloning. This can be done by using the usual procedure by the operating company in the field.

  In one embodiment, the disclosure herein provides a method of inducing an immune response against HPV infection in a subject comprising administering to the subject a novel composition comprising an HPV peptide sequence in combination with one or more adjuvants. Wherein the HPV peptide sequence corresponds to HPV16 E6 peptide and / or HPV16 E7 peptide, and the peptide sequence has a binding affinity of 5 HLA supertypes and an IC50 of about 5000 nM; Some of the peptides are multi-epitope. The methods disclosed herein for inducing an immune response can include inducing an immune response for prophylactic or therapeutic purposes. In certain embodiments, the peptides used in the novel composition are described in SEQ ID NOs: 5, 9, 10, 11, 23, 24, 25, 26, 31, 32, or herein as described, for example, in Tables 1-5. One or more peptides selected from the group consisting of others may be included. The peptides may exist as individual peptides, or a particular selected peptide may be conjugated to each other (in any order) with or without spacers, A single long peptide may be formed. The method may include the use of a composition wherein the adjuvant consists of a cationic lipid, for example, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, Includes S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations or analogs thereof. In one embodiment, a method for inducing an immune response comprises a composition comprising a peptide consisting of SEQ ID NO: 5, 9, 10, 11 and R-DOTAP, or SEQ ID NO: 23, 24, 25 or 26 and R-DOTAP. Administering to the subject a composition comprising a peptide consisting of

  The methods described herein can include the use of a novel HPV peptide composition for treating a subject having an HPV infection, wherein the infection includes, but is not limited to, common warts, plantar warts Genital, flat genital, genital genital, anal genital dysplasia, genital cancer (genital vulva, vagina, cervix, penis, anus), head and neck cancer, genital growth abnormality Infectious disease, localized epithelial hyperplasia, oral papilloma, oropharyngeal cancer, bullous cyst, and laryngeal papillomatosis.

  For example, it will be understood by those skilled in the art that changes can be made to a peptide using general molecular biology techniques, for example, by amino acid substitution, deletion, addition, and the like. In general, conservative amino acid substitutions can be applied without loss of function or immunogenicity of the polypeptide. This can be confirmed according to general procedures well known to those skilled in the art.

  The range of peptides described herein also includes peptide modifications and peptide fragments. Such modifications include, but are not limited to, substitution of natural amino acids with other molecules at specific sites, including natural and unnatural amino acids. Such substitutions can alter the physiological activity of the peptide and produce biological or pharmacological agonists or antagonists. Such substitutions may include conservative substitutions known to those skilled in the art, such as valine for alanine. Acceptable substitutions may also include modifications of amino acids such as norleucine relative to leucine. It can be appreciated that substitution of D amino acids for L amino acids is encompassed within the scope of the invention. Some substitutions are described in the Dictionary of Biochemistry and Molecular Biology, 2 "a ed., J. Stenesh, John Wiley & Sons, 1989, which is incorporated herein by reference in its entirety. Addition of amino acids such as tyrosine or other amino acids, addition of molecules such as lysine, addition of radioactive and / or non-radioactive labels at specific positions in the peptide or fragments thereof to enhance the labeling ability with radioactive and non-radioactive labels including.

Furthermore, one skilled in the art can recognize individual substitutions, deletions or additions in the amino acid sequence of the disclosed peptides or in the nucleotide sequence encoding the amino acids in the peptide, and a single amino acid in the encoded sequence or A small number of amino acid changes, additions or deletions (typically less than 5%, more typically less than 1%) are conservatively modified mutations, where the modification is a chemical modification of the amino acid. This results in a substitution with a similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art. The following six groups each contain amino acids that are conservative substitutions for each other:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), leucine (L), methionine (M); valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

Lipid adjuvants Cationic lipids have been reported to have a strong immunostimulatory adjuvant effect. The cationic lipids of the present invention can form liposomes that are optionally mixed with an antigen and can contain cationic lipids alone or in combination. Suitable cationic lipid species include: 3-β [ ⁴ N- ( ¹ N, ⁸ -Diguanidinospermidine) -carbamoyl] cholesterol (BGSC); 3-β [N, N-Diguanidinoethyl- Aminoethane) -carbamoyl] cholesterol (BGTC); N, N ¹ N ² N ³ tetra-methyltetrapalmitylspermine (cellfectin); Nt-butyl-N'-tetradecyl-3-tetradecyl-aminopropane-amidine (CLON fectin) Dimethyldioctadecylammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate) (DOSPA); 1,3-dioleoyloxy-2- (6-carboxyspermyl) -propylamide (DOSPER); 4- (2, 3-bis-palmitoyloxy-pro Pyr) -1-methyl-1H-imidazole (DPIM) N, N, N ', N'-tetramethyl-N, N'-bis (2-hydroxyethyl) -2,3 dioleoyloxy-1,4 -Butanediammonium iodide) (Tfx-50); N-1- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium chloride (DOTMA) or other N- (N, N- 1-dialkoxy) -alkyl-N, N, N-trisubstituted ammonium surfactants; 1,2 dioleoyl-3- (4'-trimethylammonio) butanol-sn-glycerol (DOBT) or cholesteryl (4'trimethyl) Ammonia) Butanoate (ChOTB) When the trimethylammonium group is bound to either a double chain (in the case of DOTB) or a cholesteryl group (in the case of ChOTB) via a butanol spacer arm; DORI (DL-1,2 -Dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylamino) (Propyl-β-hydroxyethylammonium) (DORIE) or its analogues as described in WO 93/03709; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC) Lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES) or the cationic lipids described in US Pat. Iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamide-ethylenetrimethyl Ammonium iodide, 1-dimethylamino-3-tri Methylammonio-DL-2-propyl-cholesteryl-3-β-succinate iodide, 2- (2-trimethylammonio) -ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide, 3 -β-N- (N ', N'-dimethylaminoethane) carbamoylcholesterol (DC-chol), and 3-β-N- (polyethyleneimine) -carbamoylcholesterol; O, O'-Dimyristyl-N-lysylaspal Tart (DMKE); O, O'-Dimyristyl-N-lysyl-glutamate (DMKD); 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 1,2-Dilauroyl-sn- Glycero-3-ethylsulfocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylsulfocholine (DMEPC); 1,2-dioleoyl-sn-glycero-3-ethylsulfocholine (DOEPC); 1,2-dipalmitoyl-sn-glycero-3-ethylsulfocholine (DPEPC); 1,2- Distearoyl-sn-glycero-3-ethylsulfocholine (DSEPC); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoyldimethylaminopropane (DODAP); 1,2-palmitoyl-3-trimethyl Ammonium propane (DPTAP); 1,2-distearoyl-3-trimethylammonium propane (DSTAP), 1,2-myristoyl-3-trimethylammonium propane (DMTAP); and sodium dodecyl sulfate (SDS). The present invention contemplates the use of structural variants and derivatives of the cationic lipids disclosed herein.

（式中、
R¹が、四級アンモニウム基であり、Y¹が、炭化水素鎖、エステル、ケトン、及びペプチドから選択され、R²及びR³が、飽和脂肪酸、不飽和脂肪酸、エステル結合炭化水素、リン酸ジエステル、及びそれらの組み合わせから独立に選択される）
によって表される構造を有する非ステロイド系キラルカチオン性脂質を含む。DOTAP、DMTAP、DSTAP、DPTAP、DPEPC、DSEPC、DMEPC、DLEPC、DOEPC、DMKE、DMKD、DOSPA、DOTMAは、この一般構造を有する脂質の例である。 A particular embodiment of the invention is a compound of the following formula:

(Where
R ¹ is a quaternary ammonium group, Y ¹ is selected from hydrocarbon chains, esters, ketones, and peptides, and R ² and R ³ are saturated fatty acids, unsaturated fatty acids, ester-linked hydrocarbons, phosphoric acids Independently selected from diesters and combinations thereof)
A non-steroidal chiral cationic lipid having a structure represented by: DOTAP, DMTAP, DSTAP, DPTAP, DPEPC, DSEPC, DMEPC, DLEPC, DOEPC, DMKE, DMKD, DOSPA, and DOTMA are examples of lipids having this general structure.

  In one embodiment, the chiral cationic lipid of the present invention is a lipid in which the bond between the hydrophilic group and the amino group is stable in an aqueous solution. Thus, the properties of the complexes of the present invention are their stability during storage (ie, their ability to maintain a small diameter over time and their bioactivity over time after their formation). Such bonds used for cationic lipids include amide bonds, ester bonds, ether bonds and carbamoyl bonds. One skilled in the art can readily appreciate that liposomes having more than one cationic lipid species can be used to produce the complexes of the invention. For example, liposomes containing two cationic lipid species, lysyl-phosphatidylethanolamine and β-alanyl cholesterol ester have been disclosed for specific drug delivery (Brunette, E. et al., Nucl. Acids Res., 20: 1151 (1992)).

  Considering chiral cationic liposomes suitable for use in the present invention and optionally suitable for mixing with antigens, the method of the present invention is not limited to the use of the cationic lipids described above, and cationic liposomes are produced. It can be further understood that any lipid composition can be used as long as the resulting cationic charge density is sufficient to activate and induce an immune response.

  Thus, the lipids of the present invention can include other lipids in addition to the cationic lipid. These lipids include, but are not limited to, lysophosphatidylcholine (1-oleoyl lysophosphatidylcholine), for example, lysolipids, cholesterol, or neutral phosphorus including dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). Lipids and various lipophilic surfactants containing polyethylene glycol moieties, including Tween-80 and PEG-PE are examples.

  The cationic lipids of the present invention can also include negatively charged lipids as well as cationic lipids, so long as the net charge of the formed complex is positive and / or the surface of the complex is positively charged. The negatively charged lipids of the present invention are those comprising at least one lipid species or combination thereof having a net negative charge at or near physiological pH. Suitable negatively charged lipid species include, but are not limited to, CHEMS (cholesteryl hemisuccinate), NGPE (N-glutaryl phosphatidylethanolamine), phosphatidylglycerol and phosphatidic acid or similar phospholipid analogs.

  Methods for producing liposomes used to produce the lipid-containing drug delivery complex of the present invention are well known to those skilled in the art. An overview of the methodologies for the preparation of liposomes can be found in Liposome Technology (CFC Press New York 1984); Liposomes by Ostro (Marcel Dekker, 1987); Methods Biochem Anal. 33: 337-462 (1988) And in US Pat. No. 5,283,185. Such methods include freeze-thaw extrusion and sonication. Both unilamellar liposomes (average diameter less than about 200 nm) and multilamellar liposomes (average diameter greater than about 300 nm) can be used as starting components for preparing the complexes of the invention.

  In the cationic liposomes utilized to produce the cationic lipid vaccines of the present invention, the cationic lipid is about 10 mol% to about 100 mol%, or about 20 mol% to about 80 mol% of the total liposomal lipid in the liposome. Present in mole percent. Neutral lipids, when included in liposomes, can be present at a concentration of about 0 mol% to about 90 mol%, or about 20 mol% to about 80 mol%, or 40 mol% to 80 mol% of total liposomal lipid. . When included in the liposome, the negatively charged lipid may be present at a concentration ranging from about 0 mol% to about 49 mol%, or from about 0 mol% to about 40 mol% of the total liposomal lipid. In one embodiment, the liposomes contain cationic and neutral lipids in a ratio between about 2: 8 and about 6: 4. Furthermore, it is understood that the complexes of the invention can include modified lipids, proteins, polycations or receptor ligands that function as targeting factors that direct the complex to a particular tissue or cell type. Examples of target factors include, but are not limited to, acyloglycoprotein, insulin, low density lipoprotein (LDL), folic acid and monoclonal and polyclonal antibodies against the cell surface. In addition, positive surface charges can be sterically blocked by incorporating lipophilic surfactants containing polyethylene glycol moieties to modify the circulatory half-life of the complex.

  Cationic lipid vaccines can be stored in isotonic sucrose or dextrose solutions upon recovery from the sucrose gradient, or they can be lyophilized and then reconstituted in isotonic solutions prior to use. In one embodiment, the cationic lipid complex is stored in solution. The stability of the cationic lipid complex of the present invention is measured by a specific assay to determine the physical stability and biological activity of the cationic lipid vaccine over time during storage. The physical stability of the cationic lipid vaccine is determined by quasi-elastic light scattering by methods well known to those skilled in the art, such as electron microscopy, gel filtration chromatography, or using, for example, the Coulter N4SD particle size analyzer described in the Examples. It is measured by determining the diameter and charge of the cationic lipid complex by measurement. As determined when the cationic lipid vaccine is purified, the physical stability of the cationic lipid complex is such that the diameter of the stored cationic lipid vaccine exceeds the diameter of the cationic lipid complex. , Greater than 100%, or less than 50%, or less than 30%, “not substantially change” for storage.

  While it is possible for a cationic lipid to be administered in pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation. The pharmaceutical composition using the chiral cationic lipid complex of the present invention is a physiologically compatible sterile such as phosphate buffered saline, isotonic saline, or low ionic strength buffer such as acetate or Hepes. A cationic lipid vaccine may be included in the buffer (an exemplary pH is in the range of about 5.0 to about 8.0). The chiral cationic lipid vaccine can be administered as an aerosol or as a solution for intratumoral, intraarterial, intravenous, intratracheal, intraperitoneal, subcutaneous, and intramuscular administration.

  The formulations of the present invention may incorporate any stabilizer known in the art. Exemplary stabilizers are cholesterol and other sterols that can cure the liposome bilayer and help prevent the bilayer from collapsing or destabilizing. Agents such as polyethylene glycol, poly-, and mono-saccharides can also be incorporated into liposomes to modify the liposome surface and prevent it from being destabilized due to interaction with blood components. . Other exemplary stabilizers are proteins, saccharides, inorganic acids or organic acids that can be used either alone or as a mixture.

  Many pharmaceutical methods can be used to control, modify or prolong the duration of immune stimulation. Controlled release preparations are achieved by using polymer composites such as polyesters, polyamino acids, methylcellulose, polyvinyl, poly (lactic acid) and hydrogels to encapsulate or encapsulate cationic lipids and release them slowly. obtain. Similar polymers can also be used to adsorb liposomes. To modify the release profile of the stimulant, liposomes can be included in the emulsion formulation. Alternatively, the duration of the presence of the stimulant in the blood circulation can affect the surface of the liposome, other compounds such as polyethylene glycol or other polymers, and other saccharides such as saccharides that can improve the circulation time or half-life of liposomes and emulsions. It can be improved by coating with a substance.

  When an oral formulation is required, the chiral cationic lipid can be combined with common pharmaceutical carriers known in the art such as, for example, sucrose, lactose, methylcellulose, carboxymethylcellulose, or gum arabic. Cationic lipids can also be encapsulated in capsules or tablets for systemic delivery.

  Administration of the chiral cationic lipid compositions of the present disclosure can be for either prophylactic or therapeutic purposes. When provided prophylactically, the cationic lipid is provided prior to any disease sign or symptom. When provided therapeutically, the cationic lipid is provided at or after the onset of the disease. The therapeutic administration of immunostimulatory agents helps to reduce or treat the disease. For both purposes, the cationic lipid can be administered with an additional therapeutic agent or antigen. For both purposes, the cationic lipid can be administered with an additional therapeutic agent or antigen. When a cationic lipid is administered with an additional therapeutic agent or antigen, a prophylactic or therapeutic effect can occur for a particular disease including, for example, a disease or disorder caused by HPV.

  The formulation of the present invention is a pure chiral cationic lipid as described above as a mixture of R and S enantiomers, together with one or more therapeutic ingredients such as antigens or drug molecules, for both veterinary and human use. Including only. The formulation can conveniently be provided in unit dosage form and can be prepared by any method known in the pharmaceutical industry.

Terminology The term “a” or “an” indicates one or more. As such, the terms “a” (or “an”), “one or more”, and “at least one” may be used interchangeably herein.

  The words “comprise”, “comprises” and “comprising” are to be interpreted comprehensively, not exclusively. The words “consist”, “consisting” and variations thereof are to be interpreted as exclusive rather than inclusive.

  As used herein, the term “about” means 10% variability from a given standard, unless otherwise specified.

  As used herein, the terms “subject” and “patient” are used interchangeably and include, for example, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig Or mammals such as non-human primates such as monkeys, chimpanzees, baboons or gorillas.

  As used herein, the terms “disease”, “disorder”, and “condition” are used interchangeably to indicate an abnormal condition in a subject.

  Unless defined otherwise herein, technical and chemical terms used herein are published by those of ordinary skill in the art and provide general guidance to those skilled in the art for many terms used in this application. Has the same meaning as commonly understood by reference to an object.

  The compositions of the present disclosure include a composition of an amount of HPV peptide effective to produce an immunogenic response in a subject. Specifically, the dosage of the composition to achieve a therapeutic effect depends on the formulation, the pharmacological efficacy of the composition, the patient's age, weight and sex, the condition being treated, the severity of the patient's symptoms, delivery Depends on factors such as route and patient response pattern. The treatment and dosage of the composition may be administered in unit dosage form, and one skilled in the art may consider adjusting the unit dosage form to reflect the relative activity level accordingly. The decision regarding the particular dosage used (and the number of times it is administered per day) is within the discretion of one of ordinary skill in the art and may vary depending on the dosage setting for the particular situation to produce a therapeutic effect. . Furthermore, one skilled in the art can calculate any change in the effective amount of the composition due to changes in composition components or dilution. In one embodiment, the composition can be diluted two-fold. In other embodiments, the composition may be diluted 4 times. In still other embodiments, the composition may be diluted 8 times.

  Thus, an effective amount of a composition disclosed herein can be from about 1 mg to about 1000 mg per dose based on a 70 kg mammal, eg, a human subject. In other embodiments, the therapeutically effective amount is from about 2 mg to about 250 mg per dose. In further embodiments, the therapeutically effective amount is from about 5 mg to about 100 mg per dose. In still other embodiments, the therapeutically effective amount is about 25 mg to 50 mg, about 20 mg, about 15 mg, about 10 mg, about 5 mg, about 1 mg, about 0.1 mg, about 0.01 mg, about 0.001 mg.

  Effective amounts (when administered therapeutically) can be provided on a regular schedule, ie on a daily, weekly, monthly, or yearly basis, or on an irregular schedule with varying dosing days, weeks, months, etc. Alternatively, the therapeutically effective amount to be administered can vary. In one embodiment, the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses. In other embodiments, the therapeutically effective amount of the first dose is less than the therapeutically effective amount for one or more of the subsequent doses. Equivalent dosages include, but are not limited to, about every 2 hours, every 6 hours, every 8 hours, every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 72 hours, It can be administered over various time periods, including about every week, about every 2 weeks, about every 3 weeks, about every 2 months, about every 3 months, and about every 6 months. The number and frequency of medications that correspond to the completed course of treatment can be determined according to the judgment of the healthcare professional.

  A composition can be administered by any route, taking into account the particular medical condition for which it is selected. Compositions are, among others, infusion, inhalation (including oral, intranasal, intratracheal), ophthalmic, transdermal (through a simple passive diffusion formulation, or for example using micro-needles using iontophoresis. (By enhanced delivery using poration, radiofrequency ablation or the like), intravascular, skin, subcutaneous, intramuscular, sublingual, intracranial, epidural, rectal, intravesical, and intravaginal Can be delivered to.

  The composition can be formulated neat or with one or more pharmaceutical carriers and / or excipients for administration. The amount of pharmaceutical carrier can be determined by the solubility and chemical nature of the peptide, the chosen route of administration and standard pharmacological conventions. The pharmaceutical carrier may be a liquid, and may incorporate both a solid and liquid carrier / matrix. A variety of suitable liquid carriers are known and can be readily selected by one skilled in the art. Such carriers include, for example, dimethyl sulfoxide (DMSO), saline, buffered saline, cyclodextrin, hydroxypropylcyclodextrin (HPβCD), n-dodecyl-β-D-maltoside (DDM) and mixtures thereof Can be included. Similarly, various solid (hard or soft) carriers and excipients are known to those skilled in the art.

  The composition can be administered alone, but can also be administered in the presence of one or more physiologically compatible pharmaceutical carriers. The carrier can be in dry or liquid form and must be pharmaceutically acceptable. Liquid pharmaceutical compositions can be sterile solutions or suspensions. When liquid carriers are utilized, they can be sterile liquids. Liquid carriers can be utilized in preparing solutions, suspensions, emulsions, syrups and elixirs. In one embodiment, the composition can be dissolved in a liquid carrier. In other embodiments, the composition can be suspended in a liquid carrier. One skilled in the formulation arts can select an appropriate liquid carrier depending on the route of administration. The composition can alternatively be formulated in a solid carrier. In one embodiment, the composition can be compressed into unit dosage forms, ie tablets or caplets. In other embodiments, the composition can be added to a unit dosage form, ie, a capsule. In further embodiments, the composition may be formulated for administration as a powder. A solid carrier can serve a variety of functions, i.e., serve two or more excipients as described below. For example, the solid carrier may also act as a flavoring agent, lubricant, solubilizer, suspending agent, filler, glidant, compression aid, binder, disintegrant, or encapsulating material. In one embodiment, the solid carrier acts as a lubricant, solubilizer, suspending agent, disintegrant, or encapsulating material. The composition can also be subdivided to include an appropriate amount of the composition. For example, a unit dose can be a packaged composition, such as a packaged powder, vial, ampoule, prefilled syringe, or sachet containing liquid.

  In one embodiment, the composition can be administered by a modified release delivery device. As used herein, “modified-release” refers to, for example, over a period of at least about 8 hours (eg, extended delivery) to at least 12 hours (eg, sustained delivery). Refers to controlled delivery of the disclosed composition. Such devices may also allow for immediate release (eg, therapeutic levels achieved in less than about 1 hour, or less than about 2 hours). Suitable modified release delivery devices are well known to those skilled in the art.

  Also provided are kits comprising the compositions disclosed herein. The kit may further comprise a package or container containing the composition formulated for the delivery route. Suitably, the kit includes instructions for administration and a fold for the composition.

  Numerous packages or kits are well known in the art for formulating pharmaceutical compositions for periodic use. In one embodiment, the package has an indicator for each period. In other embodiments, the package is a foil or blister package, a labeled ampoule, a vial or a bottle.

  The packaging means of the kit is that an inhaler, syringe, pipette, eye dropper, catheter, cytoscope, trocar, cannula, pressure release device, or formulation is applied to the affected area of the body such as the lung, injected into the subject, and into the bladder tissue It can be adapted for administration itself, such as other such devices that can be delivered or applied to and mixed with other components of the kit.

  One or more components of these kits can also be provided in a dry or lyophilized form. If the drug or ingredient is provided in dry form, reconstitution is generally by adding a suitable solvent. It is envisioned that the solvent can also be provided in a separate package. The kit may include means for containing the vial, or other suitable tightly enclosed packaging means for commercial use such as, for example, an injection molded or blow molded plastic container in which the vial is held. Regardless of the number or type of packages, and as described above, the kit also includes or is packaged with a separate device to assist in the injection / administration or placement of the composition into the animal body. obtain. Such devices are inhalers, syringes, pipettes, forceps, measuring spoons, eye drops, catheters, cytoscopes, trocars, cannulas, pressure delivery devices or any such medically approved delivery means. obtain.

  The terms “treat”, “treating”, or any variation thereof, are meant to include therapies utilized to ameliorate a health problem or condition in a patient or subject. In one embodiment, the health problem or condition may be resolved continuously or in a short period of time. In other embodiments, the severity of a health problem or condition, or one or more symptoms characteristic of a health problem or condition, can be reduced continuously or in a short period of time. The effectiveness of the pain treatment can be determined using any standard pain index, such as those described herein, or can be determined based on the patient's subjective pain. A patient is considered “treated” if a decrease in pain or a decrease in response to a stimulus that should cause pain is reported.

  The invention is further illustrated by the following examples, which should in no way be construed as limiting the scope of the invention. On the contrary, it can be clearly understood that other various embodiments, modifications, and equivalents may be included. This may suggest itself to those skilled in the art after reading the description herein without departing from the essence of the specification.

  Examples are provided below to assist in a complete understanding of the present invention. To date, to facilitate the development of peptide vaccines in a wide range of patients, vaccines have been developed to contain overlapping peptide sequences of 15-30 amino acids that cover the entire protein sequence of HPV. Despite the availability of in silico peptide binding assays that can determine binding peptides to various HLA molecules, this approach is naturally processed intracellularly for all peptides that can bind to MHC molecules. Is not well used in HLA-independent vaccine design because it is well established that the actual HLA range may be significantly lower than expected.

  Therefore, to successfully develop a simple HPV therapeutic vaccine that can provide broad patient coverage without using the majority of peptides and without having to fully show all E6 and E7 protein sequences in the vaccine An alternative approach was necessary. The first step in the process developed here to design simpler formulations was the design of several libraries of peptides from HPV16 E6 and E7 proteins. The next step is to screen peptides and confirm or understand the ability of the immune system to correctly process certain peptides and present the correct T epitope to CD8 + and CD4 + T cells via MHC class I and class II, respectively. To conduct extensive in vivo studies. The only way to obtain this information accurately is through actual in vivo studies to assess T cell responses.

  In this study, T cell responses were assessed by tumor regression studies using the TC-1 tumor model in C57 / B6 mice and interferon-gamma ELISPOT studies in HLA-A2 humanized transgenic mice. The next step was the selection of appropriate or preferred peptides based on the resulting T cell immune response and elimination that resulted in no or very weak T cell response. The selected active peptides were then analyzed by in silico binding analysis to determine their binding affinity for various HLA molecules. At the end of the combined analysis of in vivo data and in silico analysis, the objective is predicted by in silico analysis when used in combination, and an active peptide derived from in vivo studies that can cover at least 90% of the human population It was to select the minimum number of sequences. Finally, the selected peptides were combined into a vaccine formulation. This formulation was then tested for T cell induction potency, and competition for binding sites resulted in substantially no decrease in the potency of the selected sequence and the T cell response to all included peptides was still It was confirmed that it was obtained. Finally, the expected broad range of indication was confirmed in human clinical trials.

  Herein, sequence selection was achieved using both tumor regression studies in C57 / B6 mice and interferon-gamma ELISPOT studies in HLA-A2 humanized transgenic mice. In peptide evaluation studies conducted using tumor regression, the positive T cell response was the T cell response that resulted in tumor regression. In the HLA-A2 mouse model ELISPOT test, at least 20 spots per million splenocytes were considered positive reactions.

  Provided herein are summary results indicating positive or negative T cell responses. The details provided will enable one skilled in the art to understand the importance of extensive screening of peptide sequences by in vivo means as a first step and to follow along all steps of the present invention.

  Also provided herein are explanatory diagrams showing that peptide HLA binding prediction by in silico binding analysis confirms and corresponds to at least 90% patient coverage. These illustrations allow anyone with knowledge in the field to first select E6 and E7 peptide sequences that can be immunogenic and then test them in vivo to ensure processing and presentation. Can follow. Third, selected sequences that are shown to be appropriately immunogenic in vivo are selected and sequences that are less immunogenic are eliminated. Fourth, the peptides are subjected to in silico binding analysis to determine the binding affinity of each peptide for the various HLA types. Finally, peptide combinations that are expected to provide at least 90% coverage are selected. In the present case, human clinical trials were conducted to test and confirm the prediction.

The following examples illustrate exemplary ways of making and practicing the present invention. However, since similar results can be obtained using alternative methods, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are merely exemplary. The peptide sequences used to illustrate the present invention are provided in Table 1.

Example 1
Peptide Sequence Screening from TC-1 Tumor Regression in C57 / B6 Mice This example shows TC-1 for their effective processing and presentation of key epitopes leading to HPV specific T cell induction and subsequent tumor regression Illustrates the evaluation or screening of several HPV E7 peptides in a mouse tumor regression model.

  In the tumor regression screening test, mice were transplanted with 100,000 TC-1 cells on day 0. On day 7, once the tumor is well established, certain formulations formulated either alone or in combination with cationic lipids such as DOTMA, DOEPC or R-DOTAP to facilitate uptake and presentation Mice were vaccinated with the peptide sequence. In these studies, the vaccine contained selected peptides at a concentration of 0.1-3.0 mg / mL and cationic liposomes at a concentration of 0.2-3.0 mg / mL. When the peptide sequence was lipidated by attaching palmitoyl chains, the peptide was simply mixed with cationic liposomes prior to vaccination. In the case of non-lipidated peptides, liposomes were produced and the peptides were encapsulated by conventional thin film liposome preparation methods (F Szoka, and D Papahadjopoulos, Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes), Annual Review of Biophysics and Bioengineering, Vol. 9: 467-508, 1980). Other methods well known to those skilled in the art can also be used.

  Sample tumor regression plot showing results for peptides corresponding to SEQ ID NO: 1 (white squares), SEQ ID NO: 7, 8, 31 32 and 31 (black circles), SEQ ID NO: 7 (white circles), and SEQ ID NO: 8 (black triangles) Is shown in FIG. The result is the fact that some HPV E7 sequences resulted in effective tumor regression, but others did not, probably because of their ineffective ability to present the required CD8 + T cell epitopes. To emphasize. The results of the selection are summarized in Table 1.

Example 2
Peptide Sequence Screening by IFN-G ELISPOT Test in HLA-A2 Mice This example highlights an approach for assessing T cell responses using the ELISPOT test in humanized HLA-A2 transgenic mice. In these studies, the vaccine contained selected peptides at a concentration of 0.1-3.0 mg / mL and, when used, cationic liposomes at a concentration of 0.2-3.0 mg / mL. Humanized HLA-A2 transgenic mice with components of the human immune system capable of recognizing human antigens were vaccinated on days 0 and 7 with 100 μL of vaccine. On day 14, mice were sacrificed and HPV-specific immune responses were assessed by interferon-gamma ELISPOT (4-8 individuals per group) using standard techniques using splenocytes from the mice. Splenocytes were stimulated with specific CD8 + T cell epitopes or long multi-epitope peptides identified in Table 2. For a specific example, the number of IFN-g spots per million cells is listed. A minimum of 20 spots was a cut-off for sufficient titer and a choice for binding epitope testing. The ability of humanized transgenic mice to successfully generate a T cell immune response (> 20 spots) when stimulated with each of a long multi-epitope peptide as well as a short single-epitope peptide demonstrates that the selected peptides are effectively processed. To be presented to T cells. In these studies, unstimulated splenocytes and splenocytes stimulated with an irrelevant peptide were used together as negative controls in both assays. ConA was used as a positive control in the assay.

Example 3
The effects of chemical modifications such as oxidation and multimer formation of selected sequences on the immunogenicity of HPV16 vaccines. This study investigated the immunogenicity and their processed and presented and human CD8 + HPV specific T cell responses. This was done to evaluate the effect of chemical modification of selected peptides on their ability to stimulate. The peptides corresponding to SEQ ID NO: 23 and 25 contain cysteine, which leads to the formation of dimers and other multimers upon oxidation. Two vaccine formulations containing peptides corresponding to SEQ ID NOs: 23 and 25 were prepared.

  In formulation A: Both peptides were highly pure monomers confirmed by HPLC analysis. The vaccine contained 1.5-1.6 mg / mL of each peptide.

  In formulation B: Significant oxidation and multimer formation of both peptides was confirmed by HPLC analysis.

  Prior to vaccination, both formulations were mixed 1: 1 with 5.8 mg / mL R-DOTAP.

  The immunogenicity of the peptides was compared as described in the above examples in 10 HLA-A2 mice per formulation by assessing their ability to induce CD8 + T cells. The ELISPOT test was performed as described above. Table 3 below shows the results of the ELISPOT test and a comparison using the T test. The results of the statistical analysis show that the difference between formulation A and formulation B does not meet the p-value of P <0.05 for significance. This study suggests that modifications such as oxidation do not adversely affect the immunogenicity of the peptide sequence.

Example 4
Summary of in silico analysis of potential HLA alleles capable of binding to HPV peptides present in multi-epitope peptide vaccines containing four selected peptide sequences. Several HPV E16 and E17 peptide sequences were converted to humanized HLA- The ability to be processed efficiently in A2 mice and the specific epitopes presented to stimulate antigen-specific T cells as described in the above examples were designed and evaluated in vivo. As will be apparent to those skilled in the art, peptide epitope interactions are complex depending on a number of factors including stereochemistry, the presence of cofactors and the biochemical nature of the environment. Thus, selection of an appropriate peptide for optimal efficacy in a vaccine is neither normal nor predictable practice. Based on the appropriate peptide sequences capable of effective processing and extensive in vivo testing to identify the displayed T cell epitopes, the selected peptides have the ability to induce regression of established tumors or IFN- They were further selected based on their ability to induce gamma-induced HPV-specific T cells and then analyzed by in silico binding analysis to assess their HLA coverage. The prediction was then confirmed in human clinical trials.

  The HLA supertype, HLA-A2, accounts for about 42% of the population (Sette, A. and J. Sidney, Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics, 1999. 50 (3-4): p. 201-12). HPV16 E6 and E7 express experimentally confirmed epitopes that can be presented by HLA2 A2, as well as epitopes that can be presented by other HLA types.

  To do this, nine major HLA supertypes (Sette, A. and J. Sidney, HLA supertypes and supermotifs: a functional perspective on HLA polymorphism. Curr Opin Immunol, 1998. 10 (4): p. 478-82 Peptide binding affinities for 9 different HLA molecules representing) were evaluated. Nine major HLA supertypes account for over 98% of the peptide binding capacity of the human population (Sette, A. and J. Sidney, Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics, 1999. 50 (3-4): p. 201-12). Extensive in vivo screening tests that lead to the identification of suitable sequences for assessment of HLA binding, as well as competition for binding sites, reduces the ability of the immune system to process and present specific T cell epitopes when combined in vaccine formulations A confirmatory test to ensure that it does not occur is described in the experimental examples provided herein. This study shows that peptide selection and peptide screening techniques are available, but the process of identifying, selecting and combining peptides to optimize the process of creating an effective vaccine with broad applicability is generally Indicates that it is neither objective nor direct.

  Table 4 shows, for the purposes of this disclosure, the results of peptide binding analysis of four preferred peptide sequences identified by the T cell induction test, as well as the epitope prediction tools present in the immune epitope database (www.iedb.org). To summarize the possible epitopes covered by their sequences. This tool calculates the predicted binding affinity (IC50) between a peptide and a particular HLA class I allele. An IC50 of 5,000 nM or less is generally reported to be sufficient for biologically relevant binding and presentation with a binding affinity of less than 500 nM, which is believed to represent high affinity binding. In the current analysis, 2,000 nM was selected as a cutoff to evaluate potential HLA-binding peptides. As shown in Table 4, at least 5 different HLA molecules representing 5 different HLA supertypes are biologically significant binding of different peptides within SEQ ID NOs: 5, 9, 10, and 11 or 23-26 and It is revealed that it has the possibility of presentation. These five supertypes, regardless of ethnicity, are known to represent more than 90% of the human population (Sette, A. and J. Sidney, Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics, 1999. 50 (3-4): p. 201-12).

Example 5
Compatibility of other cationic lipids beyond R-DOTAP with HPV-E6 and E7 peptide sequences and the ability to induce HPV-specific T cell responses. Identified long HPV peptides are other cationic lipids other than DOTAP The peptide corresponding to SEQ ID NO: 26 was used as a model peptide in combination with two other cationic lipids DOTMA and DOEPC. In order to assess the ability of peptides to be effectively processed and presented in the presence of both cationic lipids, ELISPOT studies were performed using normal C57 / B6 mice. The ELISPOT test was performed as described above and was used to determine effective CD8 + T cell induction.

  In formulation 1, 1.35 mg / mL DOTMA was mixed at 1: 1 v / v with 0.5 mg / mL peptide corresponding to SEQ ID NO: 26.

  In formulation 2, 1.67 mg / mL DOEPC was mixed at 1: 1 v / v with 0.5 mg / mL peptide corresponding to SEQ ID NO: 26.

  For each formulation, 4 mice were vaccinated on days 0 and 7. On day 14, mice were sacrificed and splenocytes were used for ELISPOT testing.

  This test demonstrates that various cationic lipids can be used with the identified HPV E6 and E7 peptides to induce strong HPV-specific T cell responses.

Example 8
Use of lipidated peptides (SEQ ID NOs: 23-28) and R-DOTAP cationic lipids in HPV therapeutic vaccines and assessment of HPV-specific T cell responses in human clinical trials> 90% population based on in vivo T cell responses and peptide binding 6 peptide preparations containing 4 peptides (SEQ ID NOs 23-26) deduced in Examples 1-4 covering 2 and 2 additional single epitope peptides already contained in SEQ ID NOs 24 and 25 (sequences Nos. 27 and 28) have been evaluated in human clinical trials (clinical trials. Gov no. NCT02065973). The two single epitope peptides included in the formulation do not provide further coverage because they are already included in two of the included long peptide sequences.

  In this exploratory study, the immune response to the vaccine was evaluated by both IFN-γ and granzyme-b ELISPOT because of the diverse nature of the biological system and the response in humans. Each subject received 3 vaccinations with the R-DOTAP / peptide formulation. All subjects received 3 vaccinations once every 3 weeks. For immune monitoring by ELISPOT, blood was collected before vaccination (baseline) and 14-19 days after each vaccination and 90 days after the last vaccination. To confirm the broad coverage of the vaccine, subjects were not limited by HLA type. ELISPOT analysis was performed using the subject PBMC and stimulated with 6-peptide mixture to determine effective presentation of HPV-peptide and T cell recognition.

  The results of human clinical trials are shown in Table 6 below. A vaccine-induced response is generally defined as a 2- or 3-fold increase in immune response above baseline. In this study, the immune response was defined as a three-fold increase in post-vaccination T cell response compared to baseline samples by either IFN-γ or granzyme-b analysis. Two subjects who showed a very strong T cell response at the IFN-g spot above 420 at baseline (the immune system is probably responding to the most recent HPV infection), both subjects 2 and 5, are abnormal (Table 6). All subjects were tested for HLA type. Since HLA-A2 is the most common HLA type, half of the subjects were HLA-A2 as expected. However, all subjects, including non-HLA-A2 subjects (HLA-A1, 30, 3, 74, 80, etc.) developed a strong T cell response to the vaccine. This study confirmed that the combination of four multi-epitope peptides, SEQ ID NOs: 23-26, provides a widely applicable human HPV16 vaccine that can be recognized by patients of various genetic backgrounds.

Claims (20)

  A composition comprising an HPV peptide sequence, wherein the HPV peptide sequence corresponds to an HPV16 E6 peptide and / or HPV16 E7 peptide, and the peptide sequence binds to 5 HLA supertypes and an IC50 of about 5,000 nM A composition having affinity, as well as some of the peptides being multi-epitope peptides.   2. The composition of claim 1, wherein the peptide comprises SEQ ID NO: 5, 9, 10 and 11, or a lipidated version thereof consisting of SEQ ID NO: 23, 24, 25 and 26.   The composition of claim 2, wherein the peptides are present in the composition as individual peptides.   3. The composition of claim 2, wherein the peptides are conjugated to each other with or without a spacer to form a single long peptide comprising a sequence as claimed.   The composition of claim 1 further comprising an adjuvant.   6. The composition of claim 5, wherein the adjuvant consists of a cationic lipid.   The cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof or The composition according to claim 6, comprising an analogue.   The HLA supertype consists of HLA-A * 02: 01, HLA-A * 03: 01, HLA-A * 24: 02, HLA-B * 07: 02, and HLA-B * 58: 01 The composition according to 1.   2. The composition of claim 1, further comprising an enhancer for the epitope.   2. The composition of claim 1, further comprising a single epitope peptide.   11. The composition of claim 10, wherein the single epitope peptide comprises SEQ ID NO: 14, or SEQ ID NO: 15 and analogs thereof such as SEQ ID NOs: 27 and 28.   2. The composition of claim 1, wherein the peptide comprises modified peptides, peptide analogs, and active fragments thereof.   13. The composition of claim 12, wherein the peptide is cross-linked, PEGylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated by oxidation, disulfide bonds.   The composition according to claim 1, wherein the peptide consists of SEQ ID NO: 5, 9, 10, 11, 23, 24, 25 or 26, and the cationic lipid comprises R-DOTAP.   8. The composition of claim 7, wherein the peptide is encapsulated in a ribosome comprising a cationic lipid.   A method for inducing an immune response against HPV infection in a subject comprising administering to the subject a composition comprising an HPV peptide sequence, wherein the HPV peptide sequence corresponds to an HPV16 E6 peptide and / or an HPV E7 peptide And wherein the peptide sequence has a binding affinity of 5 HLA supertypes and an IC50 of about 5,000 nM, and some of the peptides are multi-epitope peptides.   17. The method of claim 16, wherein the peptide consists of SEQ ID NO: 5, 9, 10, 11, 23, 24, 25, or 26.   The method of claim 16, further comprising an adjuvant.   The cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof or 19. A method according to claim 18, comprising an analogue.   20. The method of claim 19, wherein the peptide consists of SEQ ID NO: 5, 9, 10, 11, 23, 24, 25 or 26 and the cationic lipid comprises R-DOTAP.

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